Alzheimer's disease (AD) is the most common form of dementia. Typically diagnosed in patients over the age of 65, an early onset form of the disease can strike much earlier. With the overall aging of the world's population, the prevalence of AD is expected to increase markedly. Unfortunately, there is no effective treatment or cure.
The extracellular deposition of amyloid peptides in the brains is thought to be a central event in AD pathogenesis. Evidence that amyloid may play an important role in early pathogenesis comes primarily from studies of individuals affected by the familial form of AD (FAD) or by Down's syndrome. The generation of amyloid-β peptide (Aβ) from amyloid precursor protein APP occurs via a regulated cascade of cleavage by at least three proteases (secretases). The recent identification of several such secretases is a major step in understanding the regulation of Aβ formation. An important AD therapeutic goal is the inhibition of these secretases. The theoretical specificity and tractability of protease targets suggest that it should be possible to generate secretase-specific protease inhibitors that penetrate the blood-brain barrier (BBB). Many studies using new knowledge of the ability of the β-secretase (BACE) to identify inhibitors by screening or rational design approaches are underway (U.S. Pat. Nos. 5,744,346; 5,942,400; 6,221,645; 6,313,268; and published PCT applications WO00/47618, WO98/21589, WO96/40885). There is no evidence for additional functions of Aβ, so there are no clear concerns about reduction of this metabolite. Two such secretases are present in many different cells in the body, and it is reasonable to assume that they have substrates besides APP. Consequently, complete inhibition of any one of these enzymes might result in toxicity, particularly during chronic treatment. At the mRNA level, BACE is expressed widely in human brain. Expression is also high in the pancreas, although enzymatic activity in this tissue is low.
Proteolytic processing of APP generates Aβ peptide, which is thought to cause the pathology and subsequent cognitive decline in AD. To initiate Aβ formation, BACE cleaves APP at the N-terminus to release sAPPB, a ˜100-kD soluble N-terminal fragment, and C99, a membrane-bound 12-kD C-terminal fragment that is cleaved by γ-secretase to generate AB peptide. The site of BACE cleavage has been determined. Cleavage by BACE between APP residues 671 and 672, which generates the N-terminal Asp residue of Aβ, is the first cleavage in the cascade that leads to mature Aβ. Extracellular release of Aβ results in the formation of amyloid plaque, while intracellular accumulation of insoluble Aβ and of other APP-derived peptide cleavage products can be toxic to cells.
One FAD family was shown to have a mutation in APP that coincided with the predicted BACE cleavage site. This “Swedish” double mutation results in overproduction of AB peptide when transfected in cells, suggesting that it is a better BACE substrate. A Met→Leu substitution at the P1 position of APP, found in the Swedish FAD mutation that causes early-onset AD, greatly enhances BACE cleavage, but many other substitutions (e.g., Met→Val) decrease BACE cleavage. These findings demonstrate the presence of a BACE activity responsible for a cleavage event that liberates the N-terminus of Aβ peptide and shows that the process is secretory not lysosomal.